FIGURE 3.

Point mutations R665W and L845F augment the responsiveness of PLCγ₂ to exogenous wild-type Rac2. A, COS-7 cells were transfected as indicated with 500 ng/well vector encoding wild-type PLCγ₂ (WT) or PLCγ₂^R665W (R665W) (left panel) or wild-type PLCγ₂ (WT) or PLCγ₂^L845F (L845F) (right panel) and increasing amounts of vector encoding Rac2. Note that the vectors encoding mutant PLCγ₂ were used at the same maximal amount (500 ng/well) in the left and right panels to observe the stimulation by wild-type Rac. Twenty four hours after transfection, the cells were incubated for 20 h with myo-[2-³H]inositol, and inositol phosphate formation was then determined. The ED₅₀ values of vector encoding Rac2 for the stimulation of mutant PLCγ₂ activity obtained by non-linear curve fitting are shown above the graphs in nanograms. B, homogenates from cells functionally analyzed in A were subjected to SDS-PAGE and immunoblotting using an antibody reactive against the c-Myc epitope.